Abstract 2876: Characterization of P2X4 purinergic receptors in the prostate tumor microenvironment
Authors: Vahinipriya Manoharan, Angelo M. De Marzo, Janielle P. Maynard
Journal: Cancer Research (2023)

Abstract The 5-year survival rate for patients with metastatic prostate cancer (PCa) in the United States is 30.6%. Therefore, more effective treatment approaches are urgently required. The P2X4 purinergic receptor is well studied for its role in inflammation. Our previous studies demonstrated that the P2X4 receptor is highly expressed in PCa compared to benign regions and is associated with poor prognosis. We observed intense P2X4 expression on most macrophages and some neutrophils in the primary tumor microenvironment (TME). Our study also showed that P2X4 knock-down in mouse PCa Myc-CaP cells resulted in significantly attenuated subcutaneous allograft growth in mice when compared to control cells. Allograft tumors were heavily infiltrated by P2X4-positive host immune cells. However, which components of the prostate TME express P2X4 receptors and the functional consequence of that expression is not well defined. Therefore, the objective of this study is to characterize P2X4 receptor expression and function in the prostate TME and metastatic niche. A P2X4 antagonist has completed Phase 1 clinical trials for the treatment of neuropathic pain. As such, deciphering a role for P2X4 in the prostate TME promises novel cancer therapeutic approaches against aggressive PCa. We used RNA in situ hybridization and immunohistochemistry to assess P2X4 expression on immune cells in human PCa tissues and Myc-CaP allograft primary and metastatic tumor tissues. To determine P2X4 function in macrophages in vitro, we used THP1 human monocytic cells differentiated to M0 macrophages and polarized to the M1 and M2 subtypes. M1 and M2 cells were treated with P2X4 agonist, CTP or antagonist, 5-BDBD. RNA was extracted from cells to examine the expression of macrophage subtype-specific genes, and cytokine levels in cell media were measured by ELISA. Statistical analysis was done using GraphPad Prism. Dual staining revealed abundant CD163+ M2 macrophages that express P2X4 in human PCa tissues. In Myc-CaP allograft primary and metastatic tumor tissues, we observed abundant F4/80+ macrophages and few Ly-6G+ neutrophils. Staining patterns suggest that P2X4+ immune cells likely include both cell types. CTP treatment of THP1 cell-derived M2 macrophages promoted anti-inflammatory IL-10 secretion while 5-BDBD treatment induced IL-6 secretion in M1 cells. M1 marker CD80, and M2 markers CD163 and CD206 mRNA expression were comparable across treatment groups, suggesting that treatment does not likely influence macrophage polarization. In conclusion, our study indicates that tumor-associated macrophages express P2X4 receptors both in primary and metastatic tumors. Our in vitro studies suggest a role for P2X4 receptors in macrophage cytokine secretion. Future studies will include functional assays to determine the role of P2X4 receptors on macrophages in the context of PCa. Citation Format: Vahinipriya Manoharan, Angelo M. De Marzo, Janielle P. Maynard. Characterization of P2X4 purinergic receptors in the prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2876.